Hepatitis C animal model

ABSTRACT

A hepatitis type C animal model into which cDNA derived from hepatitis C virus has been introduced. This animal model is useful for clarification of an onset mechanism of hepatitis C and as well as for development of means for treating the disease.

This application is a Continuation of U.S. Ser. No.: 09/214,471, filedJan. 21, 1999.

This application is a 371 of PCT/JP97/02675 filed Jul. 24, 1997.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a hepatitis C animal model, which isuseful for clarification of an onset mechanism of hepatitis C,development of means for treating the disease, and so on.

BACKGROUND OF THE INVENTION

Until now, many attempts have been made to produce an onset model ofhepatitis by introducing a hepatitis C virus (hereinafter, referred toas “HCV”) gene into a small animal such as mouse and expressing theintroduced gene (C. Pasquinelli et al., Abstract book of 2ndinternational meeting on hepatitis C virus and related viruses (Jul.31-Aug. 5, 1994 San Diego, USA); C. Pasquinelli et al., Abstract book of3rd international meeting on hepatitis C virus and related viruses (Aug.28-Sep. 3, 1995 Gold coast, Australia); Kazuhiko Koike et al., J.General Virology 76 pp.3031-3038 1995; and T. Kato, Arch Virol 141pp.951-958 1996). However, unlike many other genes, the HCV gene wasdifficult to be integrated into a murine individual, and even if it wassuccessfully integrated into the animal, production of an HCV proteindid not take place in most cases. Furthermore, even in the extremelyrare case where an HCV protein was produced in the animal, use as ahepatitis model was impossible since the protein was produced from thefetal period, thereby causing an immunological tolerance which resultedin no animal exhibiting typical hepatitis symptoms after birth.

PROBLEM TO BE SOLVED BY THE INVENTION

In clarifying an onset mechanism of a human disease and developing meansfor treating the disease, an animal model presenting pathologicalconditions very similar to those of the disease plays an important role.

However, as described above, a hepatitis C animal model has not yet beenproduced, and this has been one of the obstacles in clarifying the onsetmechanism of hepatitis C.

The present invention arises from such a technical background. An objectof the present invention is to provide a novel animal model thatexhibits the pathological conditions analogous to human hepatitis C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a structure of pCALN/pBR vector.

FIG. 2 shows sites of HCV—derived cDNA to be inserted into pCALN/pBR.

FIG. 3 is a photograph showing the results of agarose gelelectrophoresis with respect to the PCR products.

FIG. 4 is a photograph showing the results of agarose gelelectrophoresis with respect to the PCR products.

FIG. 5 is a photograph showing the results of Western blotting withrespect to proteins from the murine spleen cells.

FIG. 6 is photographs showing the results of immunofluorescence stainingwith respect to proteins from the murine fibroblast.

FIG. 7 shows changes in the serum GPT value and the HCV core protein inthe mice into which HCV-derived cDNA has been introduced, with the lapseof time.

FIG. 8 is a microscopic photograph of murine hepatic cells, beforeadministration of AxNCre, into which HCV-derived cDNA has beenintroduced.

FIG. 9 is a microscopic photograph of murine hepatic cells, 5 days afteradministration of AxNCre, into which HCV-derived cDNA has beenintroduced.

FIG. 10 is a microscopic photograph of murine hepatic cells, 7 daysafter administration of xNCre, into which HCV-derived cDNA has beenintroduced.

DESCRIPTION OF THE INVENTION

In order to solve the above-described problem, the present inventorshave studied intensively. As a result, it has now been found thatpathological conditions very similar to human hepatitis C could bedeveloped in a mouse by introducing HCV-derived cDNA into the mouse suchthat the cDNA was switch-expressed, whereby the present invention wasaccomplished.

Thus, the present invention is a hepatitis C animal model into whichcDNA derived from hepatitis C virus has been introduced.

Hereinafter, the present invention will be described in detail.

First, features of the animal model according to the present inventionwill be described.

In one example of the hepatitis C animal model of the present invention,cDNA derived from HCV is introduced into the animal such that the cDNAis switch-expressed. The term “switch expression” as used herein refersto an expression system wherein a specific gene can be expressed at adesired time. For example, the switch expression may be constructed byinterposing a sequence, which can be removed at a desired time, betweena gene to be expressed and a promoter thereof, or by using a promoterinducible with an agent or the like. An example of the former isCre/loxP expression system (Nat Sternberg et al., J. Molecular Biology150. pp.467-486, 1981; and Nat Sternberg et al., J. Molecular Biology150. pp.487-507 1981). The Cre/loxP expression system comprises aninsert gene that has been interposed between two loxP sequences, whichintervene between a promoter and a gene of interest so as to suppressexpression of the gene; and a P1 phage Cre DNA recombinase enzyme(hereinafter, simply referred to as “Cre”) which removes the insert genetogether with one of the loxP sequences. The gene of interest can beexpressed at any time by action of Cre. The loxP sequence is DNA derivedfrom the gene of E. coli P1 phage, which DNA has the followingnucleotide sequence with a length of 34 bp:ATAACTTCGTATAGCATACATTATACGAAGTTAT (SEQ ID NO: 1)

Cre is a DNA recombinase enzyme derived from E.coli P1 phage, with amolecular weight of about 38 kD.

By switch expression of the HCV-derived cDNA, a protein derived from HCVcan be produced at a stage where an animal has grown to some extent.Thus, the problem of the immunological tolerance as seen in the knownhepatitis C animal models can be avoided by the switch expression.

The outward characteristic of the hepatitis C animal model according tothe present invention is that it exhibits pathological conditionsanalogous to those of human hepatitis C. As seen in the Examplesdescribed below, examples of pathological conditions include increase inserum GPT; emergence of acidophilic body in liver tissue and exfoliationof hepatic cells; hypertrophy and hyperplasia of Kupffer's cells; andconglomerate of lymphocytes. These pathological conditions, however,vary depending on DNA fragments to be introduced, types of animal modelsand so on, and therefore they do not limit the technical scope of thepresent invention.

Next, a process for producing an animal model according to the presentinvention will be described.

The hepatitis C animal model of the present invention may be produced,for example, as follows. First, a vector in which HCV-derived cDNA isswitch-expressed is prepared. Such a vector may be prepared, forexample, from a vector including a promoter and loxP sequences, byinserting cDNA derived from HCV into a site downstream of the loxPsequence. Examples of the vector including the promoter and the loxPsequences are PCALNLW made by Yumi Kanegae and pCALN/pBR made by thepresent inventors. E.coli introduced with pCALN/pBR has been depositedwith the National Institute of Bioscience and Human-Technology Agency,Industrial Science and Technology (1-1-3, Higashi, Tsukuba-shi,Ibaragi-ken, JAPAN) under Accession No. FERM P-15753 (deposition date:Jul. 22, 1996). Examples of cDNAs to be inserted into the vectorinclude, but are not limited to, CN2, N24 and CR, which are employed inthe- Examples below. Such cDNA may be prepared according to the methodof Nobuyuki Kato et al. Proc. Natl. Acad. Sci. USA, 87, 9524-9528,1990).

An expression cassette is subsequently cut out from the prepared vector,and introduced into a fertilized egg which is then transplanted into aprovisional parent. The introduction of the vector into a fertilized eggmay be conducted according to common methods including microinjection.There is no limitation of types of animals usable as the subject forintroducing cDNA as long as they are animals where techniques ofproducing a transgenic animal have been established for them. Althoughmice are used in the Examples herein, the animals usable in the presentinvention are not limited thereto and may include rat, rabbit, pig,killifish, and zebrafish.

The animals obtained from the provisional parent are screened for thosehaving HCV-derived cDNA then for those having HCV-derived cDNA capableof the switch expression, thereby obtaining hepatitis C animal models ofthe present invention. Whether the animal has cDNA derived from HCV maybe determined by a PCR method. Specifically, DNA is extracted fromanimals to be screened and is used as a template to synthesizeoligonucleotides corresponding to both termini of the introduced cDNA.Using the oligonucleotides as primers, PCR is performed. An amplifiedfragment is detected if HCV-derived cDNA has been introduced, butundetected if HCV-derived cDNA has not been introduced. Furthermore,whether HCV-derived cDNA has been switch-expressed can be determined byremoving a sequence that inhibits the expression of the HCV-derived cDNAand then examining, either in vitro or in vivo, whether a proteincorresponding to the cDNA is produced. Whether a protein correspondingto the HCV-derived cDNA is produced may be determined by a method suchas Western blotting or immunofluorescence staining.

Since cDNA from HCV is switch-expressed, the hepatitis C animal modelaccording to the present invention can not develop hepatitis by itself.For the onset of hepatitis, it is necessary to remove a sequencesuppressing the expression of the cDNA. To remove such a suppressionsequence, a DNA recombinase enzyme may be used; for example, if thesuppression sequence is a loxP sequence then the animal cells may beinfected with an adenovirus AxNCre that expresses Cre, i.e., an enzymefor removing the loxP sequence. Cre and AxNCre can be prepared accordingto the teaching of Yumi Kanegae et al., Nucl. Acids Res. 23, 19, 3816-21, 1995.

EXAMPLES

The present invention will be described in more detail by Examples setforth below. However, its technical scope is not limited to theExamples.

Example 1

Construction of Expression Vector Utilizing Cre/loxP Expression System

An expression cassette into which a neomycin resistant gene interposedbetween two loxP sequences downstream of CAG promoter had been insertedwas cut out from PCALNLW (Yumi Kanegae et al., Nucl. Acids Res. 23, 19,38 16-21, 1995) and incorporated into pBR322 to prepare pCALN/pBR (FIG.1).

The pCALN/pBR was cleaved at a SwaI cleavage site downstream of theneomycin resistant gene and loxP sequence, into which 3 types ofHCV-derived cDNA, i.e., CN2, N24 and CR, were inserted individually(FIG. 2).

CN2 was cDNA of 3160 bp corresponding to base numbers 294-3435 of theHCV gene, N24 was cDNA of 4055 bp corresponding to base numbers 769-6823of the same, and CR was cDNA of 9162 bp corresponding to base numbers294-9455 of the same. Used as the HCV-derived cDNA were: DNAs separatedby the RT-PCR method from R6 serum which had been confirmed, by Koharaet al., to be infectious to a chimpanzee and an HPBMa cell (i.e., serumcorresponding to “plasma K” described in Yohko K. Shimizu et al., Proc.Natl. Acad. Sci. USA, 90, 6037-6041; Nobuyuki Kato et al., Proc. Natl.Acad. Sci. USA, 87, 9524-9528, 1990).

From the expression vector inserted with the HCV-derived cDNA, theexpression cassette was cut out by HindIII cleavage and then purified byagarose gel electrophoresis. The resulting DNA was further purified bycesium chloride density-gradient centrifugation. After desalting andethanol precipitation, the DNA was dissolved in TE buffer (10 mMTris-HCl, 1 mM EDTA).

Example 2

Production of Transaenic Mice

Female mice (BALB/cCrS1c and C57B1/10SnS1c, both produced by Nippon SLCCo. LTD.) were administered with an ovulatory drug and allowed to matewith male mice of the same lineage. On the following day, fertilizedeggs with pronuclei were taken out from the oviducts of the female mice.About 2 pl of the DNA fragment solution (1-5 μg/ml) prepared in Example1 was injected into the pronuclei of the fertilized eggs using a glassmicrotube. This manipulation was performed as described previously (see“Injection of DNA into fertilized egg, An Experimental Manual forEmbryological Engineering” supervised by Tatsuji Nomura and edited byMotoya Katsuki, pp. 41-76, Kodansha, 1987; and “Manipulating the MouseEmbryo, A Laboratory Manual”, B. Hogan, F. Costantini and E. Lacy,translated by Kazuya Yamauchi, Yutaka Toyoda, Yasuatu Mori, YoichiroIwakura, pp.155-173, Kindai-shuppan, 1989)

The DNA-injected fertilized eggs were transplanted into the oviducts ofpseudopregnant, provisional-parental, female mice (Slc:ICR, produced byNippon SLC Co., LTD.) which, after about 20 days, gave birth naturallyor by Caesarean section.

Example 3

Screening of Transgenic Mice

The obtained mice were screened for integration of transgenes intosomatic cell genes by PCR using primers which had sequences specific forthe HCV gene.

Auricular sections of the mice were incubated in 125 mg/ml of aproteinase K solution (50 mM KCl, 10 mM Tris-HCl pH8.3, 1.5 mM MgCl₂,0.1% gelatin, 0.45% NP40, 0.55% Tween20) at 55° C. for 1 hour and thenheated at 96° C. for 10 minutes. Five microliters of the thus-obtainedDNA solution was used for PCR. A sense primer 6-294-S20(5′TGATAGGGTGCTTGCGAGTG- 3′) (SEQ ID NO: 2) and an anti-sense primer6-604-R18 (5′-TTGCCATAGAGGGGCCAA-3′) (SEQ ID NO: 3) were used as primersfor PCR to screen mice having CN2, each primer having a finalconcentration of 1 μM. After heat treatment at 94° C. for 2 minutes, PCRwas performed using Thermal-Cylcer® (manufactured by Perkin-Elmer)through 40 cycles of reactions at 94° C. for 1 minute,

55° C. for 2 minutes and 72° C. for 2 minutes. The resultant PCR productwas subjected to 3% agarose gel electrophoresis then to ethydium bromidestaining so as to evaluate the presence or absence of the DNA product ofinterest (311 bp). Examples of the PCR products detected by the agarosegel electrophoresis are shown in FIG. 3. In the figure, A and C bothrepresent PCR products from negative mice; B represents a PCR productfrom a positive mouse; N represents a PCR product from a negativecontrol (BALB/c mouse); P represents a PCR product from a positivecontrol (pCALN-CN2 plasmid DNA); and M is a DNA size marker. Seven of 28mice obtained from BALB/cCrS1c and one of 3 mice obtained fromC57B1/10SnS1c were positive. Each positive mouse was bred for use infurther studies.

To screen the N24 gene, PCR was conducted in a similar manner to the PCRfor CN2 using a sense primer 6-4269-S20 (5′TATGACATCATAATATGTGA-3′) (SEQID NO: 4) and an anti-sense primer 6-4599-R20(5′CCCGATAATATGCTACAGCA-3′)(SEQ ID NO: 5). As a result, eight of 55 miceobtained from BALB/cCrS1c and four of 16 mice obtained fromC57B1/10SnS1c were positive.

For screening of the CR gene, the same primers as in the CN2 were used.One of 16 mice obtained from BALB/cCrS1c was positive.

Example 4

Screening for Expression in Transgenic Mice Using Culture Cells

Since the transgenic mouse according to the present invention carriesthe switch expression system, the HCV gene is not expressed in a normalstate. In order to initiate the expression, Cre must act. For thispurpose, the present inventors prepared a recombinant adenovirus AxNCrethat expressed Cre. The adenovirus AxNCre was prepared by the method ofYumi Kanegae (Nucl. Acids Res. 23, 19, 38 16-21, 1995).

Spleens were surgically excised from the transgenic mice (derived fromBALB/cCrS1c and introduced with CN2) so as to separate and culturespleen cells and fibroblasts. The cultured spleen cells and fibroblastswere infected with AxNCre at moi (multiplicity of infection) =0, 10 and100, and further cultured in 5% CO₂ at 37° C. for 48 hours. Thereafter,the cells were harvested. The thus-obtained spleen cells were collectedby centrifugation in order to extract RNA therefrom by the acidicphenol/guanidine method. Then, cDNA synthesis was conducted using theRNA as a template and 6-604-R18 (SEQ ID NO: 3) as a primer. At the endof the cDNA synthesis, 6-294-S20 (SEQ ID NO: 2) primer was added and PCRwas conducted to confirm production of HCV-derived RNA as determined bythe presence of the PCR product. According to this method, two of 2 mice(lines 8 and 13 were found to produce HCV-derived RNA (FIG. 4).

Furthermore, Western blotting and immunofluorescence staining methodswere employed in order to evaluate the expression of the HCV protein.3×10⁷ spleen cells infected with AxNCre were dissolved in 60 μl of1×Sample buffer (50 mM Tris-HCl pH6.8, 2% SDS, 10% glycerol, 5% 2ME) andheated at 100° C. for 5 minutes. The thus-obtained sample waselectrophoresed on a 10-20% gradient SDS-PAGE gel. At the end of theelectrophoresis, the resultant was transferred onto a PVDF membrane(Millipore) with a semi-dry blotting device (Millipore). The membranewas then reacted with an anti-core monoclonal antibody and an anti-E2monoclonal antibody. Two of 8 mice (lines 8 and 29) exhibited positivereactions (FIG. 5). The monoclonal antibodies used in the presentexample were prepared by producing recombinant core, E1, and E2 proteinsfrom HCV genes and then immunizing BALB/c mouse with the proteins. (seeMasahiko Kaito et al., J. General Virology 75 pp.1775-1760, 1994; andTomiko Kashiwakura et al., J. Immunological Methods 190 pp.78-89 1996)

The fibroblast cultured on slide glasses was also infected with AxNCreat moi=100, cultured for 48 hours, and thereafter fixed in a solution ofacetone:methanol=1:1 at 20° C. for 10 minutes. The fixed cells werereacted with the anti-core monoclonal antibody, the anti-El monoclonalantibody and the anti-E2 monoclonal antibody, thereby detecting the HCVproteins by the indirect immunofluorescence technique. Like the resultsof the Western blotting, two of 8 mice (lines 8 and 29) were found to bepositive (FIG. 6, line 8). Positive mice (lines 8 and 29) gave the sameresults in both of the Western blotting and immunofluorescence methods.

Example 5

In Vivo Expression of HCV Proteins

The mice that were found to be positive in expressing HCV proteins(lines 8 and 29) in the above-described cell culture experiments wereused to examine in vivo expression of the proteins. AxNCre wasadministered to the mice intravenously via the tail veins or directlyinto the spleens. After 3 days, the livers were excised from the mice toanalyze expression of HCV proteins. One milliliter of RIPA buffer (10 mMTris-HCl pH7.4, 1% SDS, 0.5% NP40, 0.15M NaCl, 1 mM PMSF) was added tothe murine liver slices, which were then homogenized at 20 strokes in aDown's homogenizer. The resultant solutions were heated at 100° C. for 5minutes and then subjected to supersonic treatment at 4° C. for 15minutes. After centrifugation at 14,000 rpm for 10 minutes, thesupernatants were separated and measured for protein concentrations.Moreover, EIA (Takeshi Tanaka et al., J. Hepatology 23:742-745, 1995)and Western blotting were conducted with respect to core proteins.

To 5 μl of each supernatant were added 50 μl of a dispersing solution(20 mM Na citrate, 0.5% NaCl, 10N Urea) for EIA of the core protein and50 μl of a denaturing solution (0.5% NaOH) for EIA of the core protein.The mixture was then allowed to react at room temperature for 10minutes. Fifty μl of a neutralizing solution (0.5M NaH₂PO₄, 5% TritonX-100) was further added for EIA reaction. The liver samples from thetransgenic mice administered with AxNCre (lines 8 and 29) were positivein EIA, whereas those from the non-transgenic mice were negative.Similarly, in Western blotting, 21 kDa of core, 37-38 kDa of E1 and67-69 kDa of E2 proteins were detected in the transgenic mice (lines 8and 29) but undetected in the non-transgenic mice.

Example 6

Liver Disorders Caused by Expression of HCV Protein in Transgenic Mice

The HCV structural proteins core, E1 and E2 were expressed in thetransgenic mice (lines 8 and 29) in order to observe changes inexpression of the HCV proteins in the livers, liver tissue images, andthe serum GPT values with the lapse of time. 10⁹ pfu of AxNCre wasadministered to the mice via their tail veins. The mice were sacrificed3, 5, 7 and 10 days after the AxNCre administration. The expression ofan HCV core protein in the transgenic mouse livers increased until Day 7and decreased on Day 10 (FIG. 7) when quantitatively measured by the EIAmethod. The serum GPT value was low until Day 5 but was drasticallyraised on Day 7, and this raising continued until Day 10 (FIG. 7).Emergence of acidophilic body and exfoliation of hepatic cells,hypertrophy and hyperplasia of Kupffer's cell, and conglomeration oflymphocytes were observed in the liver tissue from Day 3, and theysuddenly manifested themselves more strongly on Day 7 and Day 10. Inaddition to such manifestation, the regeneration image of the hepaticcells was clearly seen on Day 7 and Day 10 (FIGS. 8, 9 and 10). Thesefeatures were very much like those of human acute hepatitis, suggestingthat acute liver disorders were induced due to the expression of the HCVstructural proteins. Furthermore, in order to confirm that theacidophilic body was hepatic cells causing apoptosis, broken DNA wasstained to obtain positive results by the Tunnel method (R. Gold Lab.Invest 71, pp.219-225, 1994).

Advantage of the Invention

The present invention provides a novel human disease animal model. Thisanimal model exhibits pathological conditions very similar to humanhepatitis C and is therefore useful for clarification of an onsetmechanism of hepatitis C as well as for development of means fortreating the disease.

5 1 34 DNA E. coli P1 phage 1 ataacttcgt atagcataca ttatacgaag ttat 34 220 DNA Artificial Sequence Description of Artificial Sequence primer forPCR 2 tgatagggtg cttgcgagtg 20 3 18 DNA Artificial Sequence Descriptionof Artificial Sequence primer for PCR 3 ttgccataga ggggccaa 18 4 20 DNAArtificial Sequence primer_bind (1)..(20) Description of ArtificialSequence primer for PCR 4 tatgacatca taatatgtga 20 5 20 DNA ArtificialSequence Description of Artificial Sequence primer for PCR 5 cccgataatatgctacagca 20

What is claimed is:
 1. A transgenic mouse whose genome comprises aHepatitis C virus fragment under the control of a Cre/loxPswitch-expression system, wherein upon administration of Cre to themouse, the mouse develops at least one phenotype selected from the groupconsisting of increased serum GTP levels, emergence of acidophilicbodies in liver tissue, exfoliation of hepatic cells, hypertrophy andhyperplasia of Kupffer's cells and conglomeration of lymphocytes.
 2. Thetransgenic mouse of claim 1, wherein the Hepatitis C virus fragment isselected from the group consisting of CN2, N24 and CR.
 3. The transgemcmouse of claim 1 whose genome comprises a CAG promoter operably linkedto a cDNA sequence comprising the Hepatitis C virus fragment.